
5 

Bulletin No. 54. 

U. S. DEPARTMENT OF AGRICULTURE. 

DIVISION OK CHEMISTRY. 


T X 


5-45 



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REPORT 


AN INVESTIGATION OF ANALYTICAL METHODS FOR DISTINGUISHING 
BETWEEN THE NITROGEN OF PROTEIDS AND THAT OF 
THE SIMPLER AMIDS OR AMIDO-ACIDS. 


J. W. MALLET, 

Professor of Chemistry, University of Virginia. 

WITH 

A CHAPTER ON THE SEPARATION OF FLESH BASES FROM 
PROTEID MATTERS BY MEANS OF BROMIN. 

BY 

H. VY. WILEY, 

Chief of Division of Chemistry. 



WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 

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Bulletin No. 54. 


U. S. DEPARTMENT 

»* 

DIVISION OF 


OF AGRICULTURE. 

CHEMISTRY. 


R E P O R T 


ON 


AN INVESTIGATION OF ANALYTICAL METHODS FOR DISTINGUISHING 
BETWEEN THE NITROGEN OF PROTEIDS AND THAT Of 
THE SIMPLER AMIRS OR AMIDO-ACIDS. 


J. W. MALLET, 

Professor of Chemistry. Unicrt sity of Virginia. 

WITH 

A CHAPTER ON THE SEPARATION OF FLESH BASES FROM 
PROTEID MATTERS BY MEANS OF BROMIN. 

BY 


II. \Y. WILEY, 

CItitf of Division of Chemistry. 



WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 

1 8 9 8 . 









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LETTER OF TRANSMITTAL 


U. S. Department of Agriculture, 

Division of Chemistry, 

Washington , I). C., ’July .9, 1S9S. 

Sir: In accordance with your request of April 15, I have carefully 
read the manuscript submitted by Prof. J. W. Mallet, being a result of 
the investigation of analytical methods for distinguishing between the 
nitrogen of proteids and that of the simpler amids or amido acids. 

This investigation was undertaken by Professor Mallet at the sug¬ 
gestion of the Office of Experiment Stations, and under the immediate 
direction of the director of that oflice and of Prof. \V. (). Atwater, 
special agent. The work being purely of a chemical character, it was, 
at youi suggestion, and with the assent of Drs. True and Atwater, sub¬ 
mits d to this ofhce for inspection. The investigation consists in a 
chemical study of the methods of quantitative analysis employed in 
the separation of proteid and amid bodies, especially in animal prod¬ 
ucts. The results are similar in their scope to the chapter on this sub¬ 
ject contained in my work on the Principles and Practice of Agricultural 
Analysis, vol. 3. 

Professor Mallet has stated in an admirable manner the different 
methods which have been proposed for the separation of proteid mat¬ 
ters in animal products. By a happy modification of the phosplio- 
tungstic acid method he has greatly improved this process, and shown 
how a practical separation of the flesh bases from the other nitroge¬ 
nous substances can be effected by this reagent. The flesh bases are to 
some extent precipitated by the new form of the reagent proposed by 
Professor Mallet, but they are brought into a soluble state by the addi¬ 
tion of water and heat, so that a practically complete separation of 
them is effected. This process, together with the use of tannic acid for 
the separation of peptones, leaves little to be desired in securing a 
practically complete separation of the nitrogenous matters. The ana¬ 
lytical processes proposed have been thoroughly worked out by experi¬ 
ment upon products of known composition, so that their reliability has 
been by this means more firmly established. 

One of the most useful and simple methods of separating proteid 
matters into insoluble and gelatinoid proteids, and of separating these 
two classes from the fiesli bases, is by the use of hot water, followed by 
the use of chlorin or bromin—a method not mentioned either in the 
Principles and Practice of Agricultural Analysis, above referred to, 

3 



4 


nor in Professor Mallet’s paper. This method, which has lately come 
into use, lias been thoroughly worked out in this laboratory and applied 
in a long series of analyses of flesh products. It is so superior in every 
respect to the methods in ordinary use that it appears destined to 
entirely replace them. 1 have therefore added an outline of this method 
as an appendix to Professor Mallet’s report. 

1 submit this manuscript with the request that it be published as 
Bulletin No. 54 of the Division of Chemistry. 

I am, respectfully, 


lion. James Wilson, 

Secretary of Agriculture. 


11. W. Wiley, 

Ch ief of Dieittion. 




CONTENTS. 


REPORT BY J. W. MALLET. 

Page. 

Discussion of materials and conditions. 7 

Classes of nitrogenous constituents in food. 7 

Nutritive values of three classes. 8 

Lack of precision in chemical constitution. 8 

List of substances examined. 9 

Solutions used in experiments. 10 

Foods involved in the investigation. 11 

Methods of separating nitrogen. 11 

Dialysis . 11 

Interaction with nitrous acid. 12 

Interaction with potassium permanganate in presence of free acid or alkali. 15 

Interaction with sodium hypobromite. 10 

Behavior with cupric hydroxid (Stut/.er’s reagent). 18 

Relations to alcohol as a solvent.'. 19 

Behavior with several new or little used reagents. 19 

Behavior with phospho-tungstic acid. 19 

Classification of substances examined. 20 

. Use of hot water. 21 

Details of the method with pliospho-tungstic acid. 22 

Factors for calculation of total nitrogen. 24 

REPORT BY II. W. WILEY. 

Separation of proteid bodies from the flesh bases by means of chlorin and brornin 27 

Details of method. 27 

Factors for calculation of total nitrogen. 28 

Application to commercial meat extracts. 29 


o 




























ANALYTICAL METHODS FOR DISTINGUISHING BETWEEN 
NITROGEN OF PROTEIDS AND NITROGEN OF SIM¬ 
PLER AM IDS OR AMIDO-ACIDS. 


DISCUSSION OF MATERIALS AND CONDITIONS. 

It is admitted by all who have had experience in the chemical analy¬ 
sis of materials used as food that the common practice of determining 
the total nitrogen in such materials by multiplying the result by 6.125 
and calling the product so obtained “protein,” is but a crude and 
clumsy way of approximately representing the value of the material 
under examination as respects its nitrogenous constituents. Besides 
the substances which are properly designated as proteids there are 
other nitrogenous constituents of food materials which differ widely 
from these in nutritive value and some of which also differ greatly 
from these and from each other in the proportion of nitrogen which 
they contain. 

CLASSES OF NITROGENOUS CONSTITUENTS IN FOODS. 


The following classes of the nitrogenous constituents of food are 
commonly recognized as requiring separate consideration. 

(1) Proteids proper (by some called albuminoids), and their closely 
related derivatives, the proteoses and peptones. 

(12) Gelatinoids or collagens, and allied substances immediately 
derived from them, such as gelatin, cliondrin, etc. 

(3) Simpler amids, amido-acids, and allied substances, such as the 
asparagin, glutamin, etc., of vegetable materials, and the “ flesh 
bases” (kreatin, kreatinin, etc.) of animal origin. 

(4) Alkaloids, or amine-like compounds of welldetermined basic 
character. 

(.5) Ammonia and its salts. 

(6) Nitrates. 

To these, no doubt, should be added lecithin and analogous sub¬ 
stances containing nitrogen but closely allied to the fats. 

The known and commonly used methods for determining nitrogen in 
the forms of ammonia and nitrates, which occur but sparingly in food 
materials, may be considered fairly satisfactory. Alkaloids in the 
commonly accepted sense of the term demand attention only in con¬ 
nection with such special accessories of food as tea, coffee, and similar 
nervous stimulants, and the chief substances of alkaloidal character 
admit of being separately dealt with in these special cases without 
serious trouble and with a fair degree of accuracy. 


7 



8 


It is with tlie> first three of the above-named classes of food constit¬ 
uents that difficulty is encountered, and for which further study of 
methods is desirable. The object of the work which the writer has 
undertaken, and to which lie has devoted a good deal of time for 
several months past, has been to study the means of distinguishing 
between the first and third of these classes ot constituents, the pro- 
teids and related bodies on the one hand and the simpler amids and 
amido-acids—sometimes grouped together as ** nitrogenous extract¬ 
ives”—on the other. Incidentally only, some experiments have been 
made with representatives of the gelatinoid class. 

NUTRITIVE VALUES OK THREE CLASSES. 

It is commonly assumed that proteids, gelatinoids, and the simpler 
amids have very different nutritive values, and while all authorities 
would agree in assigning tin* highest value to the first of these there 
is probably no small difference of opinion as to the order in which the 
second and third should be rated. In considering such a question, 
there should be separately taken into account relative digestibility or 
solubility, capability of undergoing osmotic absorption, and oxidiza¬ 
bility in order to the production of energy. At present no definite 
numerical statement of the relative nutritive values of nitrogenous 
bodies of these three classes can be made. It seems much to be desired 
that more extended experiments than have so far been recorded should 
be made upon living animals—as far as possible upon human beings— 
in regard to the utilization of both the gelatinoids and the simpler 
amids. The latter no doubt undergo oxidation to some extent in the 
animal body, and produce some energy in consequence. It is probably 
true of these simpler amidic substances that much larger quantities 
than analysis exhibits as constituents of the food consumed or than 
analysis detects among the residua of food rejected from the body with¬ 
out having undergone complete oxidation, may be constantly formed 
among the earlier products of the metabolism of the proteids, and 
afterwards themselves undergo further change into the simpler and 
more stable forms of carbon dioxid, water, and urea. 

LACK OF PRECISION IN CHEMICAL CONSTITUTION. 

It must be admitted that to a chemist the question of distinguishing 
between the proteids and the simpler amids is not one of a scientifically 
precise character. The proteids doubtless contain at least a part of the 
nitrogen in the amidic relation, and where the line is to be drawn 
between more complex and more simple amids is, of course, more or less a 
matter to be arbitrarily decided. Hut, of greater importance still is the 
doubt whether any of the so-called proteids are entitled to recognition 
as definite chemical substances. We usually understand by the term 
“a definite chemical substance” a substance of which all the molecules 
are exactly alike in constitution. Thus to the chemist the identity of 


9 


such a substance as pure common salt or cane sugar or caffeine depends 
on the absolute identity in character of all the myriad molecules of 
which even the smallest sensible mass is made up. But in regard to 
such materials as these we are able to determine the relative and abso¬ 
lute number of atoms of the elements of which each molecule is com¬ 
posed, and in many cases their order of attachment to each other, or 
the “structure’’ of the molecule. On the other hand, we have reason 
to believe that the so-called proteids are made up of molecules of such 
extreme complexity, assemblages of such large numbers—hundreds— 
of atoms of carbon, hydrogen, oxygen, and nitrogen, that we can make 
but random guesses at their arrangement, and can not even determine 
with any certainty their number, relative or absolute. We talk of albu¬ 
min, myosin, syntonin, as if these terms stood for pure chemical sub¬ 
stances in the same sense that attaches to ammonia, benzene, or urea. 
But it is by no means certain that in a specimen of the most carefully 
prepared albumin from blood or white of egg any hundred, or any ten, 
molecules are absolutely alike. It may well be that a minute specimen 
of such a material consists in reality of numerous more or less similar 
but yet in some respects different molecules, which we lump together 
under a single name merely because they have a general resemblance, 
with certain properties in common. Furthermore, in nutrition investi¬ 
gations we have to deal with articles of food representing complex 
mixtures of substances referable to the two classes of the proteids and 
nitrogenous extractives, with many other things besides. Hence, an 
answer to the question to be examined must be a limited one, and such 
only as may serve the limited purpose of practical application in con¬ 
nection with nutrition investigations. Any process of separation, to be 
of value in such application, must be reasonably simple, and capable 
of being carried out without too great consumption of time. 


LIST OF SUBSTANCES EXAMINED. 


A good deal of work was necessary at the outset in procuring satis¬ 
factory specimens of the several substances to be examined. Some of 
these have been prepared out and out in this laboratory from ^natural 
animal or vegetable sources. Some have been purchased in a more or 
less crude state and carefully purified. Some have been purchased, 
and their purity ascertained by testing. 

The following representatives of the simpler amidic and imidic com¬ 
pounds were experimented with: 

Amulo-acids of the fatty series : 

Glycocin (glycocoll or amido-acetic acid). 

Alaniu (a-aiuido-propioDic acid). 

Leucin (amido-caproic arid). 

Amido-adds of the succinic acid and allied series: 

Aspartic acid (amido-surcinic acid). 

Asparagin. 

Glutamic acid (u-amido-oxyglutaric acid). 

Glutaiuin. 


10 


Jwido-acid, including a benzene nucleus: 

Tv rosin (oxy-phenyl-a-atuido-propiouic acid). 

Betaine ba*e*: 

Betaine. 

(»' uanidine has>s .* 

Kreatin. 

Kreatiniu. 

Baste and nt utral amide related to uric acid: 

Hypoxautbiue (sarkine). 

Cantina. 

Allautoine. 

As representatives of the proteijls anil allied substances tlie following 
were used: 

Allmmiu, from white of eg*;. 

Allmmin, from blood. 

Fibrin, from blood. 

Casein, front milk. 

Legumin, from peas. 

Globulin (para-glolmlin), from serum of blood. 

Yitellin, front yolk of egg. 

Myosin, from muscular tissue. 

Syntonin (acid-albumin), from muscular tissue. 

Hemoglobin (mainly oxy-ketnoglobin), front blood corpuscles. 

Albutnose, from egg albumin. 

Peptone, from fibrin of blood. 

Gelatin, front skin. 

Cbondrin, from cartilage. 


SOLUTIONS USED IN EXPERIMENTS. 


These were, for the experiments by precipitation, brought into solu¬ 
tion as follows, involving, as will be seen, change of chemical constitution 
in some cases: 

Albumin, i 

..... , , . dissolved in cold water. 

Ihemoglidmi, S 

Gelatin, t . 

l.. dissolved in hot water. 

Cbondrin, ( 

Fibrin, brought sparingly into solution by prolonged action of a 10 percent 
- solution of sodium chloride in water. 

Fibrin, digested with a 4 per cent solution of caustic soda, aud the solution 
afterwards acidified with acetic acid. 

Casein, dissolved in 1 per cent solution of caustic soda, aud the solution 
cautiously treated with dilute acetic acid to an extent just short of coagu¬ 
lation. 

Globulin, dissolved in .*» per cent solution of sodium chlorid. 

! ’ [ dissolved in 10 per cent solution of sodium chlorid. 

Myosin, S 1 

Syntonin, dissolved in 0.1 per cent solution of hydrochloric acid. 

Albumose, front the early stage of digestion of egg albumin by means of 
pepsin and 0.2 per cent solution of hydrochloric acid. 

Peptone, from advanced stage of digestion of blood fibrin by means of trypsin 
and O.2.") per cent solution of smlium carbonate. 


11 


FOODS INVOLVED IN THE INVESTIGATION. 


W liile it was especially desirable to ascertain the behavior of these 
several substances, of both classes, in their separate condition, it was 
borne in mind that in connection with nutrition investigations they 
have to be dealt with as constituents of ordinary food materials, prin¬ 
cipally the following: 


Meats, including poultry aiul fish (raw and cooked), soups and stews, meat extracts, 
eggs, milk and cheese, bread, and other farinaceous preparations such as cakes and 
pastry, fruits and raw table vegetables, cooked fruits and table vegetables, fruit- 
conserves, undigested and unabsorbed residues of food. 

It was assumed as probable that the same method (in detail) would 
uot be best adapted to all cases, but would be likely to need modifica¬ 
tion in application under different conditions. 

Of course, the investigation undertaken has gone over ground already 
well trodden, and the results recorded by Schulze, Barbieri, SachsSe, 
Kormann, Boemer, Huefner, Allen, Tankard, Chittenden, Osborne, 
Wiley, and others have been carefully examined. 

The experimental work of the present investigation is reported, not 
altogether in the order in which it was done, but rather in logical suc¬ 
cession, taking account first of physical differences between the classes 
of substances to be distinguished, then of the effects of chemical agents 
of decomposition, and, lastly, of relations to solution and precipitation. 


METHODS OF SEPARATING NITROGEN. 

A brief notice of several methods of separating nitrogen will afford 
means of comparison. The phospho tungstic method which was espe¬ 
cially investigated is treated more fully than the others. 


DIALYSIS. 

The attempt to separate such proteids as are soluble in water from 
the simpler amids intentionally mixed with them by applying Graham’s 
method of dialysis was made with no great hope of success. It is, of 
course, well known that the amorphous proteids in solution exhibit as a 
class a very small degree of diffusive mobility, while the crystallizable, 
simpler amids diff use through a porous septum, as of parchment paper, 
with much greater rapidity. But separation based on this difference 
can never be complete. Only a part of the more diffusible material 
can be obtained in the water on the opposite side of the diaphragm 
from the mixed solution, and that a large part shall be so obtained 
requires that the volume of water used shall be large as compared 
with the volume of the solution. Moreover, the absolute volume of the 
solution itself must be large where amids of but slight solubility, 
such as tyrosin, are present, in order that they may not be left behind 
in the undissolved state. But the time require^ for dialytic separation 


12 


extends to many hours, or even several days, and during such pro¬ 
tracted exposure to the air and to common atmospheric temperature 
weak solutions of the proteids readily undergo chemical change. Again, 
it is to be remembered that the peptones, classed with the proteids, are 
diffusible in much higher-degree than natural proteid material prior to 
its exposure to the action of the digestive fluids. This fact has well- 
known physiological importance in its bearing on the preparation of 
proteid food for absorption from the alimentary canal, but in analysis 
it tends to confound the particular group of the peptones with the 
simpler amids. In the recovering from weak solutions of small quan¬ 
tities of dissolved substances by the evaporation of large amounts of 
water, further chemical change of the substance recovered is likely to 
vitiate the results. 

Nevertheless, some twenty or more experiments were carried out 
with dialvsers made from the tubes of parchment paper which can now 
be bought in Germany. Cylinders of about <» inches long were cut 
from these tubes, and closed at one end by thin glass, carefully tested 
as to freedom from holes, and immersed in water contained in ordinary 
glass beakers. A fairly clean separation was obtained of leuein, aspar¬ 
tic acid, and kreatin from solutions to which lead been added egg or 
serum albumin, the diffusate being in each case evaporated at a mod¬ 
erate temperature over the water bath, and the residue weighed. But 
the process of diffusion was inconveniently slow, and less satisfactory 
results were obtained when the dialyzed sedation was made more com- 
plex by the addition of other substances. 

Subsequently the writer's attention was attracted by the paper of 
Charles.I. Martin 1 on the use for a like separation of a Pasteur filter 
in the pores of which a film of silicic acid has been deposited, the fil¬ 
tration being brought about under pressure. It has been practicable 
to make only two or three experiments in this way. The method is 
decidedly promising, but it seems more likely to be useful in the 
purification of substances in quantity than as a process of analysis. 
With small quantities of material it can hardly be made available for 
regular laboratory work in connection with nutrition investigations. 
It involves the same difficulty as any other form of dialysis in cases in 
which the proteoses and peptones are present. 


INTERACTION WITH Ni l ROUS ACII). 


It was hoped that by varying the conditions of experiment with this 
reagent some characteristic differences of behavior might be observed 
as regards the evolution of elementary nitrogen. 

The most advantageous mode of producing nitrous acid in definite 
amount was found to consist in bringing together, along with the mate¬ 
rial to be acted upon, pure silver nitrite and a hydrocldoric-acid solu¬ 
tion of known strength. The action took place in a glass flask of 


‘Jour. Physiol., 18W5, 20, pp. 







13 


about 100 c. c. capacity, closed by a stopper through which passed the 
long neck of a cylindrical funnel holding about 00 c. c., the neck hav¬ 
ing an interior diameter of but 1 mm. and separated from the wider 
cylindrical reservoir by a well ground glass stopcock. There also 
passed through the same stopper the stem of a thermometer reading 
to one-tenth of 1 degree with accuracy, a small tube with stopcock for 
the introduction of gas to displace the air of the apparatus, and a sec¬ 
ond small tube to carry off the nitrogen evolved. The capacity of the 
flask was carefully gauged with the tubes and thermometer in position 
and the stopper inserted to a marked depth in the neck, so that after 
the introduction of any known volume of solid or liquid material a 
simple subtraction would give the volume of gaseous matter remaining 
included. A gas measuring cylinder, connected with the tlask by a 
tube of very small bore and immersed in water in a larger cylinder 
which could be raised or lowered, provided lbr collecting and measur¬ 
ing the evolved nitrogen. The tlask was supported in a water bath, 
so that it could be heated or cooled at pleasure. 

In order to give time for action upon the organic material under 
experiment, and to keep down to conveniently small limits the evo¬ 
lution of nitrogen dioxid, it was found important to work with the 
nitrous acid in a sutiiciently diluted condition. With the same object 
in view it was found desirable to raise the temperature of the tlask 
very gradually. In carrying out an experiment the particular amidic 
or proteid material was finely pulverized, weighed off, and introduced 
into the flask, either in the dry state or with as little recently boiled 
water as possible, and with the necessary amount of silver nitrite, as 
a tine crystalline powder. This powder was contained in a small, thin 
glass cylinder which could be easily upset in order to mix its contents 
with the other materials in the flask. A current of nitrogen gas was 
next run through the tlask until all air was expelled. In the case of 
substances dissolving in water with ditticulty the flask was now allowed 
to stand closed for some time to permit of solution taking place. Com¬ 
munication was established with the gas-measuring cylinder and the 
proper quantity of standard hydrochloric acid made by diluting very 
strong aqueous acid with boiled water and kept in small well-closed 
bottles, was gradually introduced through a funnel with capillary bore. 
Care was taken that no air was admitted. After all action in the cold 
had ceased the temperature of the flask was gradually raised by means 
of the water bath up to a maximum of about 1M3° or 97° C. As soon as 
all action was over and the apparatus had cooled down to atmospheric 
temperature the flask was nearly tilled up with recently boiled water, 
introduced through the capillary funnel, and a small measured volume 
of nitrogen was run in to displace any remaining nitrogen dioxid. 
The gas in the measuring cylinder was then treated with oxygen in 
sufficient excess to dispose of all nitrogen dioxid and with caustic 
potash and pyrogallol to remove excess of oxygen. Alter standing at 


14 


rest for sometime t lie gas was measured, duo allowance being made for 
the nitrogen present in the Hast at first and for the small additional 
amount afterwards introduced. Of course, the proper corrections for 
pressure, temperature, and tension of aqueous vapor were made in con¬ 
nection with all the measurements of gas, and the quantity of nitrogen 
obtained was divided by 2, one half being yielded by the organic sub¬ 
stance and the other by the nitrous acid. 

With a view to guard against the retention of any nitrogen in the 
form of di-azo derivatives from amids, a moderate excess of hydro¬ 
chloric acid was used. In the case of kreatinin the results are com¬ 
plicated by the formation of the supposed nitroso compounds observed 
by Dessaignes. With several of the protcids the bright yellow color 
of the so-called xantho proteic acid was well marked, as the conse¬ 
quence, doubtless, of the action in the flask of nitric acid produced by 
the breaking up of nitrous acid into nitric acid, nitrogen dioxid and 
water. Too little is known of the yellow substance formed, Mulder’s 
xantho-proteic acid, to indicate how far its nitrogen comes from the 
proteid and how far from the nitric acid, or in what direction or to 
what extent its formation may influence the amount of elementary 
nitrogen given oft*. In the case of proteid solutions coagulable by heat 
it was manifestly important to allow the action of the hydrochloric acid 
on the silver nitrite to become complete below the temperature of 
coagulation, as otherwise silver nitrite was entangled in clots of the 
coagulating proteid. In some, at least, of these experiments, it seemed 
probable that a part of the nitrogen dioxid given off, and perhaps of 
the free nitrogen, was due to the reducing action of the carbon or 
hydrogen of the organic substance upon nitrous or nitric acid, and not 
solely to the normal interaction of nitrous acid and the amidogen radi¬ 
cle present. 

The process was varied, not only in respect to the temperature 
applied, but also, within moderate limits, in respect to the pressure on 
raising or lowering the gas-measuring tube in the water. It was further 
varied by increasing the proportion of nitrous acid to different extents 
in excess of the theoretically necessary amount. 

In all some fifty or sixty experiments were made. In a number of 
cases the simpler amidic compounds gave a fair approximation to the 
quantity of nitrogen theoretically to be expected from them, though 
even with such substances as leucin, asparagin, and aspartic acid the 
results were not as sharp as the claims of Sachsse and Kormann would 
lead one to suspect. One of the best experiments yielded for aspartic 
acid 9.57 per cent of nitrogen, instead of the 10.53 per cent actually 
present. Kreatin did not give one-half of its nitrogen, as has been 
heretofore recorded as the fact, but a somewhat nearer approach to 
one-third—10.19 per cent as against 32.00 per cent—the probability of 
which result is indicated by the accepted structure of the molecule. 
In all cases the proteids gave some nitrogen, but the proportion was 


15 


much smaller than from the simpler amids, and varied much more with 
the precise conditions of temperature, pressure, and strength of solu¬ 
tion used. In one experiment serum albumen gave but 2.G8 per cent, 
and in another but 2.92 per cent was obtained from haemoglobin. 

On the whole, the indications pointed to the simpler amids and 
amido-acids being most easily decomposed by nitrous acid, the proteoses 
and peptones perhaps next, and the proteids proper least. But no 
differences were observed upon which any useful analytical process ot 
separation or distinction could be based. Experiments made in this 
way are, moreover, troublesome, and require strict observance of the 
necessary precautions to aVoid error from introduction of air into the 
apparatus and its action on nitrogen dioxid. 

INTERACTION WITH POTASSIUM PERMANGANATE IN PRESENCE OF 

FREE ACID OR ALKALI. 

The writer had not much hope of obtaining useful results by this 
method, in view of former experience gained in connection with the 
extended research upon the determination of organic matter in drink¬ 
ing water carried out many years ago for the U. S. National Board of 
Health, of which the results were published in the annual report of 
that board for 1882. Still, as the work then done was upon extremely 
dilute solutions, comparable in respect to the amount of organic mat¬ 
ter present with natural potable waters, it seemed possible that results 
not altogether of the same sort might be obtained with solutions of 
greater concentration. Hence some thirty or forty experiments were 
made with permanganate strongly acidified with sulphuric acid, and a 
rather larger number with the same salt after potassium hydroxid 
had been added in about the same proportion as is usual for the Wank- 
lyn, Chapman, and Smith so-called albuminoid ammonia process. In 
the former set of experiments the amount of oxygen withdrawn from 
the permanganate, and in the latter set the amount of ammonia pro¬ 
duced, were determined. In both cases the reactions were carried out 
at various temperatures up to the boiling points of the several liquids. 

Some difference was observable in the results obtained with the com¬ 
paratively strong solutions used, such difference being more notable 
for alkaline than for acid permanganate. But in the main these results 
only confirmed the conclusions arrived at in the earlier research The 
effect of the reagent employed is imperfect, and varies much with 
the nature of the individual organic materials tested; much, also, 
with the conditions of the experiment and with the rate at which 
fhe action proceeds. No valid evidence is obtained in support of 
Wanklyn’s view that simple and definite fractions of the total nitrogen 
present are evolved as ammonia on treatment with alkaline perman¬ 
ganate. 

Some special difficulties and sources of error already known were 
reobserved; as, for instance, the continuous evolution of ammonia by 


16 

boiling many organic snbstauces with alkaline permanganate until dis¬ 
tillation has practically brought the contents of the retort to dryness, 
without all of the nitrogen present having been given off. And some 
other causes of trouble were noticed; as, for instance, the loss of oxygen 
given off as such from a strong and acid solution of permanganate on 
standing in a heated condition irrespective of action on the organic 
matter present. 

The general tenor of the results in the case of the nitrogenous sub¬ 
stances treated pointed to more energetic and extensive action of per¬ 
manganate in alkaline than in acid solution; also to more extensive 
action on the simpler amidic compounds tlian on the proteids. Hut 
notwithstanding sundry variations of method as to strength of the 
reagent solutions, proportion of reagent to organic material acted on, 
temperature and time of action, no indication was obtained of any 
valid basis for distinction in analysis between the two classes of 
nitrogenous material studied. 


INTERACTION WITH SODIUM IIYI’OIJROMITE. 

This reaction is so frequently used for the approximate determina¬ 
tion of urea (carbamid), while its results with other ainids have been 
so scantily examined and almost no facts bearing on its relation to the 
proteids have been recorded that a good deal of interest was felt in 
examining it somewhat extensively with the two classes of materials 
which were studied. 

The solution of bromin was prepared with 240 grams of potassium 
bromid, 200 grams of free bromin, and enough water to make up a 
liter. The solution of caustic soda was made with 340 grams of pure 
sodium hydroxid to the liter. These solutions were preserved in sep¬ 
arate bottles and equal volumes of the two were mixed just before 
using. W hen undiluted the mixture represented 0.1 gram of originally 
free bromin to each cubic centimeter and was in most of the experi¬ 
ments used of this strength; but various dilutions were also employed, 
being made by additions of water in definite amount. 

The apparatus with which the reaction was carried out was essen¬ 
tially the same as that adopted for the experiments with nitrous acid, 
save that the small tube was omitted which was intended to introduce 
an inert gas to displace air from the apparatus, this precaution being 
unnecessary in the hypobromite experiments. 

Nearly 80 experiments were made, varying the conditions as to tem¬ 
perature from that of the atmosphere, usually 15° to 20°, up to IMP to 
98° C., as to pressure within the limits allowed by the immersion of the 
gas measuring cylinder, and as to time from fifteen or twenty minutes 
up to live or six hours. 

In some cases the results obtained were in agreement with those of 
the few hitherto recorded experiments—as, for instance, aspartic acid 
gave no nitrogen, as was found to be the fact by Allen and Tankard. 


i 


17 


In other cases there was disagreement with some of tin* published 
statements and confirmation of others. Thus, Alien and Tankard 
obtained no nitrogen from glycocin, 1 and Tankard none from asparagiu, 1 
while Oeclisner de Coninck reported both these substances as acted on 
(by sodium hypochlorite) when gently heated, nitrogen gas being- 
evolved.- The writer obtained from glycocin 4.2 per cent of nitrogen 
instead of 18.07 per cent as required by the formula, 3 and from aspar¬ 
agin 11.12 per cent instead of 18.67 per cent, the amount calculated, 
taking account of the presence of a molecule of water of crystallization. 

In a number of cases the quantity of nitrogen evolved from an amid 
or amido-acid of known constitution seemed to represent a simple frac¬ 
tion of the total quantity contained in the material operated on, and it 
might not unnaturally be suspected, as in some of the cases reported 
by Allen and Tankard, that one-fourth or one-half to two-thirds of the 
whole quantity was liberated. Thus in one experiment leucin gave 2.58 
per cent of nitrogen instead of 10.09 (calculated), or about one-fourth ; in 
another, kreatin gave 21.90 per cent instead of 32.06 (calculated), about 
two-thirds; and in another, hypoxanthine gave 18.80 per cent instead of 
41.18 (calculated), which might mean one half-—two atoms out of four. 
But it is not believed there is any more real significance in these approxi¬ 
mations to definite fractional parts of the nitrogen being evolved than 
in the similar approximations to which Wanklyn drew attention in con¬ 
nection with his so-called albuminoid-ammonia process. His conclu¬ 
sions have been shown to be entirely erroneous. Different figures could 
be obtained from the same materials acted on by varying the conditions 
of the experiment, and in some cases there was no really sharp ending to 
the reaction, traces of gas continuing to be slowly given off for hours 
after the main portion had been collected. Moreover, while our knowl¬ 
edge of the constitution of kreatin would make it not improbable that 
two atoms of nitrogen out of three should be liberated, or two out of four 
in the case of hypoxanthin, there is no similar ground for any expecta¬ 
tion that leucin, containing but one atom of nitrogen in the molecule, 
should yield one-fourth; or glutamic acid, also with but one atom, should 
yield something like one-fourth (2.50 per cent instead of 9.52 calculated). 

The researches of S. Hoogewerff and W. A. van Dorp have shown 
that numerous definite products other than elementary nitrogen may 
be formed by the action of alkalin hypobromites upon amids and amido- 
acids, especially those containing cyclic nuclei. In several of the 
writer’s experiments—as, for instance, with alanin among the simpler 


'A. H. Allen, Commercial Orgauic Analysis, 1896, Vol. Ill, Part III, p. 275. Pos¬ 
sibly tin* solutions were not heated. 

'•Comptes rendii8, 1895, 121, 893-894. Jour. Chem. Soc. (London), (1896), 
Abstracts, org. chem., p. 282. 

'In Watt’s Dictionary of Chemistry, revised edition by M. M. Pattison Muir and 
H. F. Morley, Vol. II, p. 627, it is stated, on the authority of Deniges (Comptes ren- 
dus, 107, 662), that with sodium hypobromito nitrogen is evolved from glycocin. 

2925—No. 54-2 





18 


amido-acids, and with a sample of globulin among the proteids—a crys* 
talline residue separated out in tlie tlask on cooling. I his residue was 
not examined further than to determine qualitatively that it contained 
nitrogen. The proportion borne by the nitrogen left in such tixed 
residual products to that collected as gas evidently varied with the 
conditions of the particular experiment, as well as with the nature of 
the substance acted on. 


All the proteids and analogous materials treated gave some gaseous 
nitrogen. For the most part the amount was about two-fifths of the 
whole amount present; but occasionally a larger proportion, as in one 
experiment with globulin just about one-lialf, and in another with 
myosin about three fourths. In this last ease the action was allowed 
to go on at a high temperature for a time much longer than usual. 
The remarks already made in regard to the simpler amidic substances, 
as to modification of results by variation of the conditions of experi¬ 
ments, fully apply also to the experiments with proteids and their 
congeners. The lack of sharpness of ending to the reaction was more 
noticeable with the latter class of materials than with the former. 

Although these experiments with alkaline hypobromite were interest¬ 
ing, and occasionally offered points which might repay further investi¬ 
gation, they did not, taken altogether, afford any satisfactory basis for 
distinguishing in analysis between the classes of materials to be studied 
in contrast with each other. 


BEHAVIOR WITH CI PRIC HYDROXID (STUTZER’S REAGENT). 

The formation of insoluble compounds of the proteids with cupric 
hydroxid, while leaving the simpler amids soluble in the presence of an 
excess of this reagent, has been extensively adopted as the means of 
separation, but experiments made in this way have not given the writer 
much confidence in the method as a general one. In some cases, working 
with a proteid alone, the copper compound underwent partial solution, 
a blue liquid being formed, although care had been taken to avoid the 
presence of free alkali. Possibly this result was due to incipient 
decomposition of the proteid material. As Stutzcr himself has pointed 
out, peptones are very incompletely precipitated by cupric hydroxid. 
A further objection is to be found in the very slight solubility of the 
copper salts of some of the simpler amido-acids, especially leucin and 
glutamic acid; in a less degree the same statement applies to aspartic 
acid. Even at the temperature of boiling water the copper compounds 
of these substances are but very sparingly soluble, and if the liquid, 
after digestion with cupric hydroxid, be filtered cold, 1 the compounds 
in question will, if present, be almost certainly left on the filter along 
with the proteid material. 

'Ah directed by the Association of Ortioial Agricultural Chemists, Bulletin No. 4t» 
of the U. S. Department of Agriculture, Division of Chemistry (1895), p. 25. 



19 


RELATIONS TO ALCOHOL AS A SOLVENT. 


It lias been repeatedly proposed to use strong alcohol for the precip¬ 
itation of the proteids, with a view to tlieir quantitative determination, 
and this even in cases involving the simultaneous presence of some of 
the amidic compounds under discussion, such as the flesh bases. 1 But 
not only do the character and amount of the proteids so precipitated 
or left insoluble vary with the strength of the alcohol and the quantity 
of it used, but the further serious objection presents itself that nearly 
all the simpler amids and amido acids are either insoluble in alcohol or 
so slightly soluble that it is practically impossible to wash them out 
satisfactorily from the precipitated or coagulated proteids. A method 
which is not properly applicable to such important substances as 
asparagin among vegetable food materials, and kreatin among those 
of animal origin, manifestly deserves but little consideration. 


BEHAVIOR WITH SEVERAL NEW 


OR LITTLE USED REAGENTS. 


A number of miscellaneous experiments were tried with reagents 
which have either been but occasionally applied to materials of the 
kind under examination, or have not been so applied at all, so far as 
published records show. 

A weak solution of pure phenol (carbolic acid), trichloracetic acid, 
formic aldehyde in aqueous solution, and hydrazoic acid (azoimide) 
were thus tried, but from none of these reagents were results obtained 
which furnished any ground for a general method of distinguishing 
the two classes of nitrogenous materials which were being studied. 


BEHAVIOR WITH PIIOSPIIO TUNGSTIC ACID. 


This reagent, the discussion of which I have left to the last, has 
proved of much more value than any other I have tried, and its appli¬ 
cation under proper conditions affords, 1 believe, a fairly satisfactory 
practical solution of the question I have undertaken to examine. The 
use of phospho-tungstic acid for the precipitation in general of nitrog¬ 
enous compounds, alkaloidal, amidic, and proteid, is, of course, well 
known and often piacticed, but some of the special facts on which may 
be founded its application to the purpose now under discussion are 
believed to be new, and the particular use made of these points ot 
behavior has not been before described. In connection with the experi¬ 
ments made with phospho-tungstic acid, the results obtainable from a 
parallel series of experiments with a strong solution of tannic acid were 
compared, one of these two reagents being found under special circum¬ 
stances to replace the other with advantage. 

The precipitant was employed not as a sodium or other salt, but as 
the jdiospho-duodeci-tungstic acid, crystallized in small cubes and dis 


1 See Watts's 
Wiley’s Princip 


Dictionary of Chemistry, revised edition. Vol. IN', p. 330, and II 
lea and Practice of Agricultural Analysis, Vol. Ill, p. 453. 


W 


20 


solved in dilute hydrochloric acid, 25 grains of real I1C1 to tlie liter. 
Solutions of two degrees of strength were prepared, the one containing 
grams of the solid reagent to the liter, the other 100 grams. In the 
experiments with tannic acid, solutions in like manner containing ’>0 
and l(to grams, respectively, of a remarkably good sample of the 
reagent, dissolving readily to a perfectly clear liquid, were made use of. 

It has been assumed by Stutzer and others that the proteid and 
allied substances are precipitated by phospho tungstic acid, while the 
simpler amids and ainido acids are not so precipitated. As qualifying 
this general assumption, it has been stated that some ot the proteid 
derivatives, as the peptones, 1 are incompletely precipitated, and on the 
other hand that the tlesh bases, kreatin, kreatinin, etc., are fully pre¬ 
cipitated. The reagent in question has been recommended as the 
means of separating and determining them. 2 

Account does not seem to have been taken hitherto of the fact that 
some of the precipitates formed by substances of amidic character with 
phosplio tungstic acid are to a small extent soluble in water, and that 
their solubility is much increased by rise of temperature. 


CLASSIFICATION OF SUIISTANCKS KXAMINKI). 

It has been found that the various substances on which these experi¬ 
ments have been made fall into three classes, as follows: 

(a) Those which, even in pretty strong solutions, give no precipitate 
with phospho-tungstic acid. 

(/>) Those which are precipitated at any rate in strong solutions, the 
precipitate redissolving with more or less ease on heat being applied to 
the liquid or on treating the precipitate with hot water, and reappear¬ 
ing on cooling. 

(c) Those which are precipitated, the precipitate not being sensibly 
soluble and the supernatant liquid remaining clear on being heated 
along with the precipitate and subsequently cooled. 

ruder the first head fall glycocin, alanin, leucin, aspartic acid, aspar- 
agin, glutamic acid, tyrosin,and allautoin. In the case of alanin there 
was a very slight turbidity, not increased by using a saturated solution, 
suggesting the probability of a trace of some impurity being present. 

Under the second head were observed glutamiu, a slight precipitate, 
the solution easily cleared by heating, the turbidity reappearing on 
cooling; betaine in strong solution, a copious white precipitate, dis¬ 
solving gradually on addition of more water and heating, the precipi¬ 
tate reappearing on cooling; kreatin, strong precipitate, solution 

1 Dr. W. D. Halliburton in the article “Proteids*' in Watts's Dictionary of Chem¬ 
istry, revised edition, Vol. IV, p. 331. In Gamgee’a Text-hook of Physiological 
Chemistry, Vol. II, p. 139, it is stated that peptones are precipitated by phospho- 
tnngstic and phospho-molyhdic acids, and that these two reagents furnish tin* means 
of separating them. A similar ninpialitied statement is to he found in the Appendix 
(by A. Sheridan Lea) to Michael Foster’s Text-book of Physiology, p. 4f>. 

•Koenigand ltoemer—Zeitschrift fiiranalyt. Chemie.,34,560, adopted in Prof. H.W. 
Wiley’s Principles and Practice of Agricultural Analysis, 3. 454. 


21 


cleared by hearing-, becoming turbid again on cooling; kreatinin, large 
precipitate, disappearing on free addition of water and heating, reap¬ 
pearing on cooling; hypoxantkine, strong precipitate, cleared up on 
heating, reappearing on cooling; and carnine, well marked precipitate, 
cleared by moderate addition of water and heating, reappearing on 
cooling. Urea also, which is not likely to occur among food materials, 
but possibly needs to be considered in connection with undigested 
residua, gave a copious white precipitate of crystalline character, cleared 
by heating, and the precipitate forming anew on cooling. A peptone 
solution gave an abundant precipitate, becoming clotted by heating 
and dissolving to a considerable extent, reprecipitating on cooling. 

Under the third head were found egg albumin, fibrin, casein, legumin, 
globulin, vitellin, myosin, syntonin, haunoglobin, albumose, gelatin, and 
chondrin. In nearly all these cases the precipitate formed was bulky, 
taking into account the strength of the solution used, and became clot¬ 
ted on heating, shrinking very considerably. In the case of myosin 
only (in 10 per cent sodium chlorid solution) was there a very slight 
appearance of turbidity on cooling the solution which had been heated 
with the precipitate. 

USE OF HOT WATER. 


As it was evidently important to ascertain with some degree of defi¬ 
niteness how far the precipitates formed by amidic substances of the 
second of these classes would dissolve in hot water, quantitative experi¬ 
ments were made with those which seemed to be least soluble. In each 
case the precipitate formed by phospho-tungstic acid in the cold was 
filtered off, washed with cold water, and dried at ordinary temperature 
(15° to 20°) over sulphuric acid. Stutzer advises that the phospho- 
tungstic acid precipitates be washed with dilute sulphuric acid, and 
Wiley recommends for the same purpose a solution of the precipitates. 
Of the precipitate formed by betaine, 1 part dissolved in 71 parts of 
water at 98.2°; of that produced by kreatin, 1 1 part dissolved in 107 
of water at 98.1°; of that produced by kreatinin, 1 part dissolved m 
222 of water at 97.9°; of that produced by bypoxanthin, 1 part dis¬ 
solved in 98 of water at 97.6°; and of that produced by carnin, 1 part 
required for solution 182 of water at 98.4°. 

By the use of phospho-tungstic acid as a precipitant, therefore, fol¬ 
lowed by washing of the precipitate with hot water, it seems possible 
to effect a separation of all the simpler amidic substances from all the 
proteids and proteid-like bodies, except only the peptones. As regards 
this last group it is stated unreservedly by A. 8. Lea, 2 A. Gamgee, 3 


'The phospho-tungstic acid precipitate formed by kreatin, white at first, dark¬ 
ened notably on exposure to light, looking after a while like silver chloride which 
had been in like manner exposed. The experiment on solubility was made with a 
sample which had been screened from light and was unaltered. 

-The Chemical Basis of the Animal Body, an appendix to M. Foster’s Text-book of 
Physiology (1893), p.45. 

3 A Text-book of the Physiological Chemistry of the Animal Body (1893), 2, 139. 




ami W. 1>. Halliburton 1 that the peptones are precipitated by tannic 
acid. The last-named writer says u completely precipitated.” In one or 
two of the writer's own experiments, using tannic acid, an abundant pre¬ 
cipitate was formed. This became clotted on heating and the clear 
supernatant liquid showed some little return of turbidity on cooling. 
The writer is inclined, however, to attribute this apparent partial re so¬ 
lution of the precipitate merely to the presence of a little of a proteose 
formed in the earlier stages of digestion and not afterwards completely 
removed. Assuming this view to be correct, tannic acid furnishes the 
reagent needed to dispose of the one case unprovided for by phospho- 
tungstic acid. 

DKTAILS OK TilK METHOD WITH J’llOSPHO-TUNUSTlC ACID. 

The method proposed is as given in the following paragraphs. It is 
stated, for the sake of simplicity, first, as applicable to meat, raw or 
cooked. The variations required in the examination of other classes of 
food materials are reserved for notice afterwards. 

A carefully selected and accurately weighed sample is to be ground 
in a glazed porcelain mortar with as much sharp edged siliceous sand, 
previously heated to redness with free exposure to air, or with as much 
hard glass in small, sharp splinters similarly ignited, as shall suffice to 
thoroughly subdivide the tissue and reduce it to the condition of a 
smooth pulp. Of this pulp, very carefully mixed, so as to insure uni¬ 
formity, two aliquot parts are to be taken. In one the total nitrogen is 
to be determined by the well-known Kjeldahl process with addition of 
potassium sulphate, as recommended by Gunning, using a rather large 
proportion of sulphuric acid, so that no previous drying of the sample 
is needed. The other part is to*be digested with cold water, filtered on 
a nitrogen-free filter,* and the residue washed on the filter with water 
at the same low temperature as long as it gives up soluble matter in 
sensible amount. Cold water is used to avoid action on and extraction 
of the gelatinoids. Kreatinin is quite easily dissolved, as is also sarco- 
sine; kreatin with a very fair degree of ease. Xanthin, liypoxanthin, 
and earn in are less soluble. 3 

The filtrate is then to be slightly acidified with acetic acid, heated 
to about 90° O., and again filtered from any coagulum produced. A 
little more sand or pulverized glass may with advantage be stirred in 
before bringing it onto the filter the second time. 

To this second filtrate is to be added an acidified solution of phospho- 
tungstic acid as long as a precipitate continues to form, avoiding any 
very large excess of the reagent solution. With a moderate amount of 

1 Watt’s Dictionary of Chemistry, revised edition, 181*4, 4, 331. 

The rase with which filtration may be effected is much increased by the presence 
of the sand or crushed glass. 

Hypoxantbine, 1 part in 300 of water. The solubility of carnine does not seem 
to have been recorded till now. The writer bus found it to be 1 part in 312 of water 
at 15.3 C. 


23 


sand or pulverized glass added, to prevent the formation of a dense clot, 
the liquid and precipitate are to be heated to about 90° C , filtered, and 
the precipitate washed thoroughly on the filter with water at about tlie 
same temperature. This third filtration may be carried out on the 
same filter already used for the second, but as a general rule it will be 
found better to use a new filter, thus avoiding possible delay due to 
partial drying of the previously used one and subsequent clogging of 
its pores. 

Assuming now that nitrogen is present in the sample under exami¬ 
nation only in the two forms of proteids and simpler amidic compounds, 
the three (or two) filters used and their contents are to be submitted 
to the Gunning-Kjeldahl process for the determination of proteid nitro¬ 
gen. By subtraction of this from the total nitrogen previously deter¬ 
mined the amount of this element present in the simpler amidic 
compounds will be obtained. 

In cases involving the presence of ammonia or its salts, nitrates, or 
alkaloids, the nitrogen occurring in these forms must, of course, also be 
deducted from the total nitrogen before recording the residue as nitro¬ 
gen of the simpler amids and amido-acids. In like manner a separa¬ 
tion of lecithin, when present, may be effected by the use of ether as a 
solvent, 1 a determination of phosphorus made the basis of a calcula¬ 
tion of lecithin nitrogen, and this in turn subtracted from the total 
nitrogen found. 

When peptones are present, these are to be precipitated by tannic 
acid from the solution which has been acidified with acetic acid and 
heated. After this has completely cooled down, and before adding 
pliosplio tungstic acid, the filter on which the tannic-acid precipitate 
is collected and washed with cold water is, with its contents, to be 
submitted to the modified Kjeldahl process, and the nitrogen obtained 
counted as part of the proteid nitrogen. 

The several filters and precipitates from which the proteid nitrogen 
is obtained may either be treated separately by the Kjeldahl process 
or, preferably, may all be brought together and submitted to this pro¬ 
cess in a single operation. If the latter course be pursued, it will be 
well to introduce each filter with its contents as soon as washed into 
the strong sulphuric acid, so as to avoid any possible decomposition 
and loss of nitrogen as ammonia until all the filters have been brought 
together and the moist combustion process can be proceeded with. 

When proteoses are present it may be well to make a check determi¬ 
nation of their amount by saturation of the aqueous solution, after 
acidification with acetic; acid, heating and subsequent cooling, with zinc 
sulphate, 2 and determining nitrogen in the precipitate so formed by 
means of the Kjeldahl process. 


•Extraction with the ether alone will remove only a portion of the lecithins. A 
mixture of ether and alcohol should follow the ether in order to secure a complete 

extraction. 

^ As suggested by Hoenier, Zcitschrilt fur analyt. ( hemic, lS!t>, 34,<>hJ. 




24 


When gelatinoids are present, as may be the ease with soups, stews, 
and meat extracts, hot water may be used at once for solution or wash 
1 ug the original material, and this with the advantage of facilitating 
the extraction of the less soluble simpler amids and amido acids. 
These are, as a rule, more easily dissolved in the presence of a little 
free acid; hence acidification at an early stage of the treatment is 
advantageous. In a case in which tyrosin might be present, as in some 
vegetable materials, and possibly among unabsorbed residua of food, 
the use of hot water and the presence of free acid would greatly 
increase the solubility of this substance. 

In food of vegetable origin where much starch is present it will be 
better to avoid the use of hot water at first, so that the solution may 
not be loaded with viscid material, rendering filtration difficult. 

In all cases in which the food material to be examined is already 
fluid from the presence of water—as. for instance, soup, milk, and the 
like—filtration will of course at once be resorted to, being almost always 
much facilitated by the addition of sand or pulverized glass, and only 
such further quantity of water will be used as is required for washing 
the undissolved matter left upon the filter. 

In the presence of fat in large quantity, it may be well first to 
remove this, or most of it, by extraction with ether. The simpler 
atnidic substances are, as a rule, insoluble in ether, but by way of pre¬ 
caution the ethereal solution of fats might be shaken up two or three 
times with acidified water, and the watery fluid evaporated and tested 
for nitrogen. 

In regard to the method of reporting results, the most important 
point is the separate statement of the amount of nitrogen present in 
the form of proteids and their more closely related congeners and in 
the form of the simpler amids and amido-acids. But in attempting to 
calculate from the nitrogen found under these heads the actual amount 
of the proximate nitrogenous col stitucnts of the food material ex¬ 
amined, the question arises, What factor should lx* used by which to 
multiply the nitrogen found in each case? 




FACTORS FOR CALCULATION OF TOTAL NITROGEN. 

The error noticed by Professor Wiley 1 as involved in the multiplica¬ 
tion of the total nitrogen of a sample of meat by 6.25, and the assump¬ 
tion that the product represents the true quantity of nitrogenous matter, 
is not restricted to the use of the same factor for the proteids and flesh 
bases. While the multiplier should be a much smaller one for the lat¬ 
ter, it also confounds under a single head these two classes of material, 
unquestionably possessing very different nutritive values. 

It is evident that for each substance examined, or at any rate foi 
each class of generally similar food materials, there should be made a 
qualitative investigation of the simpler amidie constituents present. 

* Principle* and Practice of Agricultural Analysis (1X97), 3, 551. 



25 


and if possible a roughly approximate estimate of the proportions in 
which they severally occur; also it is clear that the factor to be used 
in calculating the nitrogenous constituents to be reported under each 
analysis should be decided by such preliminary investigation. In the 
light of present knowledge of this kind the writer is inclined to sug¬ 
gest the following numbers: 

For proteids and allied substances, multiply nitrogen found by G.25, 
as usual at present. 

For flesh bases and simpler amids of animal origin in food mate¬ 
rials, multiply by 3.05. 

For simpler amids and ainido-acids of vegetable origin in food mate¬ 
rials, multiply by 5.15. 

For mixed amidic constituents of unabsorbed solid residua in diges¬ 
tion experiments, multiply by 9.45. 

As a matter of general practice, in all statements of the results of 
nutrition experiments the rule should be invariably observed to give 
the actual amounts of nitrogen obtained by analysis, whatever calcu¬ 
lated conclusions be afterwards deduced therefrom; so that, with fur¬ 
ther knowledge of the nature of the proximate nitrogenous constitu¬ 
ents present, the factor used in calculation may be changed, if such 
change seems to be called for, while the original experimental work 
still retains its value. 

In concluding this report the writer wishes to express the hope that 
the method suggested, which seems to carry with it some improvement 
upon present practice and in a fairly simple and easily applied form, 
may be tried with yet other amids and proteids than those experi¬ 
mented on by him, and that any special difficulties which may be en¬ 
countered with particular articles of food may be investigated. Espe¬ 
cially is it desirable that the variations be studied which may prove to 
be necessary in dealing with vegetable instead of animal materials. 
The latter have been chiefly kept in view, in accordance with the 
instructions of the letter of authorization under which this investigation 
has been conducted. 

























SEPARATION OF 1ROTEID BODIES FROM THE FLESH BASES BY 

MEANS OF CHLORIN AND BROMIN. 


By H. W. Wiley. 

In dry, finely-ground animal matters from which the fats have been 
thoroughly extracted with ether, it is possible to effect an easy separa¬ 
tion of the nitrogenous bodies into three groups. These groups, for 
purposes of dietetic study, are sufficiently distinct to afford a safe basis 
of valuation of the different nitrogenous constituents. The process 
which has been adopted in the laboratory of the Division of Chemistry, 
Department of Agriculture, for this separation is given in detail here. 


DETAILS OF METHOD. 

-n the dry, fat-free, finely-ground animal substance the nitrogenous 
bodies soluble in water may be separated by first thoroughly exhaust¬ 
ing the material with cold or lukewarm water, and afterwards with 
water near the boiling temperature. By this method the water-soluble 
constituents of the nitrogenous substances are thoroughly removed. 
Having determined the total percentage of nitrogen m the whole 
sample, the residual insoluble nitrogen is determined in the residue left 
after extraction. This percentage multiplied by 6.25 gives the total 
quantity of insoluble proteid matter contained in the animal material. 
In the filtrate the soluble proteid matter which has been dissolved by 
the water may be completely thrown out of solution by treatment with 
bromin in the manner about to be described. 

About one-gram portions of the dry animal material are washed with 
ether by decantation, using from 50 to 100 cc of ether for each sample, 
and decanting the ether through filters which are afterwards used to 
receive the portion of the sample insoluble in hot water. After allow¬ 
ing the ether to evaporate, the samples are treated first with cold and 
then with hot water, this washing also being by decantation, the total 
amount of water used being from 300 to 400 cc. The undissolved resi¬ 
dues are brought on to the filter with the last portions of water. The 
nitrogen in the residues on the filters is determined by the Gunning 
method. 

The filtrate from the insoluble portions of the meat is received in 
Kjeldahl flasks and used for the separation of the soluble proteid nitro¬ 
gen by bromin. The filtrate is first acidulated with two or three drops 
of strong hydrochloric acid and then about 2 cc of liquid bromin are 
added and the contents of the flask vigorously shaken. If the bromin be 
all taken up more is added until finally a globule ot .1 <*c ot liquid bromin 

is left undissolved and the supernatant liquid is thoroughly saturated 

27 


28 


with bromin. The mixture is then allowed to stand overnight, by 
which time tlu* precipitate will have settled. 1 he supernatant liquor 
is passed through the filter and the precipitate in the flask washed by 
decantation with water, the globule of undissolved broniin serving to 
saturate the wash water so that it is unnecessary to use additional 
brouiin water for the washing. The filter containing the precipitate is 
returned to the same flask in which the precipitation has taken place 
and the nitrogen therein determined by the Guiming method, i he 
sum of the nitrogen in the part insoluble in water and the part precipi¬ 
tated by broinin is subtracted from the total nitrogen determined on 
the original sample, and the difference gives the total nitrogen m the 
flesh bases. 


FACTORS FOR CALCULATION OF TOTAL NITROGEN. 


The factors used for calculating the total nitrogenous bodies are as 
follows: 

For the part insoluble in water, >' x 0.25. 

For the part soluble in water and precipitated by bromin, N x 0.25. 

For the flesh bases, N x 3.12. 

This method is based upon investigations reported by It ideal and 
Stewart 1 last year. 

These writers recall some of the experiments made in 187b, in which 
it was shown that a current of chlorin gas, conducted through an 
aqueous solution of proteid matters, produces a precipitate which is 
of a quite constant composition, and one which can be collected, dried 
in vacuo, and weighed. They describe particularly the use of this 
reagent in precipitating gelatin prepared from the high-grade com¬ 
mercial article. They show that the total quantity of gelatin can be 
accounted for from the weight of the precipitate by multiplying the 
weight of the precipitate obtained by the factor 0.78. The authors 
also point out the possibility of using bromin for chlorin for the pre¬ 
cipitation, and state that the studies of the use of bromin are under 
way. They call attention to the fact that as early as 1840 chlorin 
had been used by Mulder for the precipitation of soluble proteids, and 
refer to a paper of his published in Berzelius’ Jahresbericht, volume 
19, page 734, in which he states results on precipitation quite similar to 
those secured by Bideal and Stewart. 

At the close of their paper Bideal and Stewart mention the work in 
this direction of I>e Vrij, Ann. 1'li.irm. 61, 248; Th£nard, Mem. 
d’Arcueil, 2, 38; Mulder, Bulletin en NVerlande, 1839, 153, and Ber¬ 
zelius’ Jahresbericht, 19, 729, on the same subject. 

Allen and Searle, 2 acting on the suggestion of Bideal and Stewart, 
worked out the bromin method by applying it to various soluble pro¬ 
teids, including the whole range from albumin to peptone. In the 

‘The Analyst, 18!»7, 22. 228 et »eq. 4 The Analyst, 18i>7, 22, 258-263. 



29 


application of this test to commercial gelatin the following process was 
employed. 

Fifty grams of commercial gelatin were dissolved in warm water and 
the solution diluted to half a liter. In 10 cc of this solution, corre¬ 
sponding to 1 grain of the gelatin, the nitrogen is determined directly 
by the Gunning- Kjeldahl process. Another portion of 10 cc is treated 
with an excess of bromin in the following manner. 

The solution is first brought to a volume of 100 cc with water and 
placed in a conical beaker with a sutlicient quantity of hydrochloric 
acid to produce distinct acid reaction. A saturated solution of bromin 
water is added in considerable excess, and the liquid stirred vigorously 
for sometime. The precipitate which separates is tiocculent when first 
formed, but becomes more viscous after stirring and adheres for the 
most part to the sides of the beaker, which, with its contents, is allowed 
to stand for about half an hour, or until all the precipitate is settled. 
The supernatant liquor is decanted through an asbestus filter. The 
precipitate adhering to the beaker is washed several times with cold, 
distilled water and the washings poured through the filter. Occasion¬ 
ally, when most of the free bromin is washed out of the precipitate, the 
liquid does not filter clear. It is therefore advisable to keep the wash¬ 
ings separated from the filtrate, and, if necessary, wash with sodium- 
sulphate solution or with bromin water. The nitrogen in the precipitate 
is determined by the Gunning-Kjeldahl process as follows: 

The precipitate which has been collected on the asbestus filter, together 
with the asbestus, is returned to the beaker in which the precipitation 
took place. Twenty cubic centimeters of strong sulphuric acid are 
added, the beaker covered with a watch glass and placed on a wire 
gauze over a lamp. When frothing has ceased, about 10 grams of pow¬ 
dered potassium sulphate are added and the liquid boiled until color¬ 
less. After cooling it is distilled with water and the ammonia distilled 
off and determined in the usual way. The percentage of nitrogen found, 
when multiplied by the factor 6.A>, or in the case of gelatin by 5.5, gives 
the amount of proteid matter precipitated by bromin. In the commer¬ 
cial gelatin above mentioned the nitrogen content was found to be 14.1 
and 14 per cent, respectively, on two determinations. Solutions of 
kreatinin, asparagin. and aspartic acid were found to yield no precipi¬ 
tates with bromin, but bromin was found to precipitate all albumin, 
acid albumin, and all peptones formed by the digestion of albumin with 
pepsin. 

APPLICATION TO COMMERCIAL MEAT EXTRACTS. 


On applying the bromin method to commercial meat extracts the 
following results were obtained. The solutions of the Bovril prepara¬ 
tions were not previously filtered, and therefore the figures contain the 
nitrogen and the fiber present. 


30 


Xitrogen in commercial meat cxtract$. 


Nitrogen 

in precip- Proieiiln 
itati* liy (N x 0.25). 
I.romin. 


/Vr cent. I'er rent. 


Lieiiic ('omnanv'H extract. 1- <1 

Smtuneil liuvrif........*. 1.94 12. 2n 

lkivril for invalid*... 2.64 16. 71 


Koenig and Roomer have shown that the proteid nitrogen in meat 
extracts is generally much overestimated. They found a total of 1.17 
per cent of proteid nitrogen in the Liebig Company’s extract, which is 
equivalent to 7.41 percent of total proteids, mostly albumose. The fact 
that bromin completely precipitates all proteid and gelatinoid matters 
in solution, affords a convenient means of solving certain problems which 
have hitherto presented considerable difficulty. For instance, in a solu¬ 
tion which has been subjected to digestion it may be possible to precipi¬ 
tate all the unchanged proteids by saturation with zinc sulphate. The 
peptones which have been formed during digestion remain in solution 
and can be separated by filtration. In the filtrate the peptones can be 
completely precipitated by bromin, and thus the total quantity of these 
bodies formed during digestion can be accurately determined. 

Allen and Searle applied this method to an examination of the Lie 
big Company’s extract, 5 grains of which were dissolved in 100 cc of 
water and the solution saturated with zinc sulphate. After filtering, 
bromin water was added to the filtrate and a precipitate produced which 
redissolved on diluting with water and the addition of hydrochloric 
acid. When the filtrate from the saturated zinc sulphate was previ¬ 
ously diluted with water and acidulated, no precipitate was formed 
on the addition of bromin. This reaction shows that no considerable 
quantities of real peptones exist in Liebig’s extract. 

Since this bulletin was prepared for the press, an extensive article 
on the halogen derivatives of albumin has been published by F. Blum 
and \V. Vaubel 1 of Frankfurt, Germany. 

1 Ueber Halogeneiweissderivate, Journal fiir praktieclie Clieniie, 1897, 56, 393-6, 
ami 1*98, 57, 365-396. 


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